Regulation of Cotton Fiber Growth by Extracellular Nucleotides and Ectoapyrases

ABSTRACT

The present invention includes compositions and methods of modulating the length of one or more cotton fibers in a plant by contacting the plant or tissue derived therefrom with at least one of: a nucleotide; a modulator of ectoapyrase gene transcription; or an anti-ectoapyrase antibody or fragments thereof, at a concentration that modulates growth of one or more cotton fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/120,273, filed Dec. 5, 2008, the contents of which isincorporated by reference herein in its entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support under Contract No.IOB-0344221 and IOS-0718890 awarded by the NSF. The government hascertain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

None.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

None.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of plant fibergrowth, and more particularly, to the regulation of plant fiber growthby modulating ectoapyrase activity or gene transcription.

BACKGROUND ART

Without limiting the scope of the invention, its background is describedin connection with cotton fiber production. Cotton fibers are some ofthe longest single cells in the plant kingdom and are considered a modelsystem for studying cell growth and primary cell wall deposition. Uplandcotton, Gossypium hirsutum, is a widely studied species that has beenused for numerous genetic and physiological studies on fiber growth(Wilkins and Arpat, 2005; Shi et al. 2006; Chen et al., 2007; Gao et al.2007; Lee et al., 2007; Taliercio and Boykin, 2007). Cotton fibersdifferentiate from the epidermis of the ovule and then undergo cellexpansion during the elongation phase of growth from 3 d post-anthesis(dpa) to 16 dpa (Tiwari and Wilkins, 1995; Basra and Malik, 1984) andgrow via diffuse growth, which does not share the common ultrastructuralcharacteristics found in tip-growing cells (Tiwari and Wilkins, 1995).

Fiber growth can be conveniently studied in cultured ovules, which allowfor testing the effects of various growth regulators on the initiationand elongation processes. For example, Shi et al. (2006) documented therole of ethylene in regulating the growth of cotton fibers in ovuleculture, and Qin et al. (2007) showed that very-long-chain fatty acidscould promote cotton fiber cell elongation in ovule culture byactivating ethylene synthesis.

DISCLOSURE OF THE INVENTION

In one embodiment, the present invention includes compositions andmethods of modulating plant fiber growth comprising: contacting a plantcell with one or more extracellular exogenous nucleotides selected fromdi-nucleotides, tri-nucleotides, or poorly-hydrolyzable nucleotides at aconcentration that modulates growth of one or more cotton fibers orcotton fiber cells. In one aspect, the one or more poorly-hydrolyzablenucleotides comprise thio, methylene, amide or methyl-modified ATP, ADP,UTP, UDP, CTP, CDP, TTP, TDP, GTP, GDP, dATP, dADP, dUTP, dUDP, dCTP,dCDP, dTTP, dTDP, dGTP, dGDP, ATPγS, ADPβS, analogues and combinationsthereof. In another aspect, the one or more extracellular nucleotidesare provided in a concentration that increases growth of the one or morecotton fibers. In another aspect, the one or more extracellularnucleotides are provided in a concentration that decreases growth of theone or more cotton fibers. In another aspect, the one or moreextracellular nucleotides are provided at a concentration of between 1μM and 100 μM to increase the growth of the one or more cotton fibers;between 125 μM and 200 μM to decrease the growth of the one or morecotton fibers; or between 10 μM and 75 μM to increase the growth of theone or more cotton fibers. In yet another aspect, the one or moreextracellular nucleotides are provided at a concentration of 5, 10, 20,30, 40, 50, 60, 70, 80, 90 or 100 μM that increases the growth of theone or more cotton fibers. In another aspect, the one or moreextracellular nucleotides are provided at a concentration of 125 μM orgreater to decrease the growth of the one or more cotton fibers. Inanother aspect, the one or more extracellular nucleotides alter theactivity of one or more ectoapyrase enzymes by altering thetranscription of the ectoapyrase gene(s).

In another embodiment, the present invention includes compositions andmethods of modulating plant fiber growth by contacting a plant cell withone or more modulators of ectoapyrase gene transcription, wherein themodulation alters the length of one or more cotton fibers. In oneaspect, the one or more modulators of ectoapyrase gene transcriptionalters ectoapyrase gene transcription are selected from anti-sense orsiRNA gene inhibitors. In another aspect, the one or more modulators ofectoapyrase gene transcription are antagonists of ectoapyrase genetranscription to decrease fiber growth. In yet another aspect, the oneor more modulators of ectoapyrase gene transcription are agonists ofectoapyrase gene transcription to increase fiber growth.

In another embodiment, the present invention includes a recombinantplant comprising a plant cell that has increased expression ofectoapyrases that increases cotton fiber growth. In another embodiment,the present invention also includes a method of modulating plant fibergrowth comprising: contacting a plant cell with one or more inhibitorsof ectoapyrase activity comprising an anti-ectoapyrase antibody orfragments thereof, wherein the inhibition decreases the length of one ormore cotton fibers.

Yet another embodiment of the present invention includes a compositionthat modulates the length of one or more cotton fibers in a plantcomprising at least one of a poorly-hydrolyzable nucleotide, a modulatorof ectoapyrase gene transcription or an anti-ectoapyrase antibody orfragments thereof, at a concentration that modulates growth of one ormore cotton fibers. In one aspect, the one or more poorly-hydrolyzablenucleotides comprise thio, methylene, amide or methyl-modified ATP, ADP,UTP, UDP, CTP, CDP, TTP, TDP, GTP, GDP, dATP, dADP, dUTP, dUDP, dCTP,dCDP, dTTP, dTDP, dGTP, dGDP, ATPγS, ADPIβS, analogues and combinationsthereof. In another aspect, the one or more extracellular nucleotidesare provided in a concentration that increases growth of the one or morecotton fibers.

For example, the one or more extracellular nucleotides are provided in aconcentration that decreases growth of the one or more cotton fibers. Inanother aspect, the one or more extracellular nucleotides are providedat a concentration of between 1 μM and 100 μM to increase the growth ofthe one or more cotton fibers; between 125 μM and 200 μM to decrease thegrowth of the one or more cotton fibers; or between 10 μM and 75 μM toincrease the growth of the one or more cotton fibers. In yet anotheraspect, the one or more extracellular nucleotides are provided at aconcentration of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μM thatincreases the growth of the one or more cotton fibers. In anotheraspect, the one or more extracellular nucleotides are provided at aconcentration of 125 μM or greater to decrease the growth of the one ormore cotton fibers. In another aspect, the one or more extracellularnucleotides alter the activity of one or more ectoapyrase enzymes.

Yet another embodiment of the present invention is a recombinant plantexhibiting increased cotton fiber growth as compared to thecorresponding wild-type plant, wherein the recombinant plant comprises arecombinant nucleic acid encoding an ectoapyrase gene inhibitor operablyassociated with a regulatory sequence. In one aspect, the regulatorysequence is a promoter, a constitutive promoter or an induciblepromoter. In another aspect, the nucleic acid is contained within aT-DNA derived vector. In another embodiment, the recombinant planttissue is derived from the recombinant plant exhibiting increased cottonfiber growth. In another embodiment, the invention is a recombinant seedis derived from the recombinant plant exhibiting increased cotton fibergrowth.

Yet another embodiment of the present invention is a method of making arecombinant plant exhibiting increased cotton fiber growth as comparedto the corresponding wild-type plant comprising: contacting plant cellswith a nucleic acid encoding an inhibitor of an ectoapyrase, wherein thenucleic acid is operably associated with a regulatory sequence to obtaintransformed plant cells; producing plants from the transformed plantcells; and selecting a plant exhibiting the increased cotton fibergrowth and yield. In one embodiment, the recombinant plant tissue isderived from the recombinant plant exhibiting increased cotton fibergrowth. In another aspect, the recombinant seed is derived from therecombinant plant exhibiting increased cotton fiber growth.

DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1. GhAPY1 is expressed in cotton fibers during elongation phase ofgrowth. [A] Quantitative RT-PCR analysis of GhAPY1 expression shows thatexpression of this apyrase is fiber specific and is rapidly up-regulatedduring in vitro fiber growth. [B] Immunoblot analysis using anti-apyraseAPY1 and APY2 antibodies confirms that there is an immunodectableapyrase in growing cotton fibers.

FIG. 2. Inhibition of apyrase activity in cotton ovule cultures usingchemical inhibitors decreases overall fiber growth and increases eATPlevels. [A] Treatment of cotton ovule cultures with varyingconcentrations (μg/mL) apyrase inhibitors NGXT 191 and apyrase inhibitor4 at 3 dpa and 5 dpa decreases average fiber lengths at 7 dpa. [B]Inhibition of fiber growth induced by application of apyrase inhibitorsNGXT 191 and apyrase inhibitor 4 (5 μm/mL) at 3 dpa and 5 dpa isreversed by co-incubation with 250 μM PPADS. [C] Treatment of cottonovule cultures with varying concentrations (μg/mL) apyrase inhibitorsNGXT 191 and apyrase inhibitor 4 at 3 dpa and 5 dpa increases the amountof ATP surrounding the cotton fibers. Different letters above the barsindicate mean values that are significantly different from one another(p<0.05; n≧24).

FIG. 3. Inhibition of apyrase activity in cotton ovule cultures usingapyrase antibodies decreases overall fiber growth and increases eATPlevels. [A] Treatment of cotton ovule cultures with polyclonalanti-apyrase antibodies at 3 dpa and 5 dpa decreases average fiberlengths at 7 dpa. The difference in growth of fibers treated withpre-immune serum was not statistically different (p>0.05; n≧24); thedifference in average fiber lengths treated with immune serum and oftubes treated with buffer is statistically significant (p<10⁻⁹; n inevery case≧20). The protein concentration of the pre-immune sera was 0.3μg/μL, and of the immune sera was 0.4 μg/μL. [B] Treatment of cottonovule cultures with polyclonal anti-apyrase antibodies at 3 dpa and 5dpa increases the amount of ATP surrounding the cotton fibers. Differentletters above the bars indicate mean values that are significantlydifferent from one another (p<0.05; n≧24).

FIG. 4. High concentrations of ATPγS and ADPβS decrease overall cottonfiber growth. [A] Representative image of effects of application of 150μM ATPγS or 150 μM ATPγS+250 μM PPADS on cotton fiber growth. [B]Application of 150 μM ATPγS and ADPβS to cotton ovule cultures.Different letters above the bars indicate mean values that aresignificantly different from one another (p<0.05; n≧24).

FIG. 5. Low concentrations of ATPγS and ADPβS increases overall cottonfiber growth. [A] Representative image of effects of application of 30μM ATPγS or 30 μM ATPγS+250 μM PPADS on cotton fiber growth. [B]Application of 30 μM ATPγS or ADPβS to cotton ovule cultures. Differentletters above the bars indicate mean values that are significantlydifferent from one another (p<0.05; n≧24).

FIG. 6. Biphasic dose response curve for ATPγS and ADPβS.

DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

Ectoapyrases (NTPDases) are enzymes that remove the terminal phosphatefrom extracellular nucleoside tri- and diphosphates. In Arabidopsis, twoectoapyrases, AtAPY1 and AtAPY2, have been implicated as key modulatorsof growth based on genetic and inhibitor studies and on the expressionpatterns of their genes. Fibers of cotton (Gossypum hirsutum) stronglyexpress an apyrase, GhAPY1, whose amino acid sequence closely resemblesthe two Arabidopsis ectoapyrases. Message and protein levels for GhAPY1are upregulated when fibers enter their rapid growth phase. In an ovuleculture system, fibers release ATP as they grow, and when their apyraseactivity is blocked by the addition of polyclonal anti-apyraseantibodies or by small molecule inhibitors, the medium ATP level risesand fiber growth is suppressed. High concentrations of the poorlyhydrolysable nucleotides ATPγS and ADPIβS applied to the medium inhibitfiber growth, and low concentrations of them stimulate growth. Treatmentof cotton ovule cultures with AMPS causes no change in the growth rateof cotton fibers. Both the inhibition and stimulation of growth can beblocked by PPADS and RB2, two antagonists that block purinoceptors inanimal cells, and by the feedback inhibitor, adenosine. These dataindicate that ectoapyrases and extracellular nucleotides play asignificant role in regulating cotton fiber growth.

Ectoapyrases (ecto-NTPDases) are well characterized in animal cells,where they play a key role in reducing the concentration ofextracellular nucleotides (e.g., eATP and eADP), which function assignaling agents to activate purinoceptors and induce diversephysiological responses, ranging from neurotransmission to programmedcell death (Burnstock, 2008; Zebisch and Strater, 2008). In Arabidopsis,two ectoapyrase enzymes, AtAPY1 and AtAPY2, play a critical role incontrolling the growth of all Arabidopsis tissues tested thus far (Wu etal., 2007). More recently Riewe et al. (2008) showed that apyrases couldregulate potato tuber growth. In Arabidopsis, these enzymes are moststrongly expressed in rapidly growing tissues, such as etiolatedhypocotyls, pollen tubes, and the elongation zone of roots (Wu et al.,2007).

Kim et al. (2006) used a recombinant hybrid reporter protein (luciferasewith a cellulose binding domain attached) to visualize ATP in the ECM ofplant cells and observed that the highest levels of ATP are foundoutside of actively growing plant cells like root hairs. These resultssuggest that plant cells, like animal cells, release ATP into theextracellular matrix (ECM) via fusion of secretory vesicles to theplasma membrane during the growth of plant cells.

Extracellular ATP and the ectoapyrases that limit its concentration canregulate growth and diverse other responses in plants (Roux andSteinebrunner, 2007). These findings led us to hypothesize thatectoapyrase activity might influence the growth of cotton fibers. Inthis study it was found that the expression of the cotton apyrase thatmost resembles AtAPY1 and AtAPY2 is highest during the rapid growthphase of the fibers, and that inhibition of apyrase activity by theaddition of chemical apyrase inhibitors or anti-APY1/APY2 Arabidopsisantibodies to cotton ovule cultures can inhibit cotton fiber growth.Moreover, applied nucleotides can promote or inhibit cotton fiber growthin a dose-dependent manner, and these effects are blocked by the sameantagonists of purinoceptors that block the effects of extracellularnucleotides in animals. Taken together, our results support the novelconclusion that extracellular nucleotides and ectoapyrases play animportant regulatory role during cotton fiber growth.

As used herein, the phrase “transgenic DNA construct” refers to asegment of DNA that is introduced into the genome of a parental cottonline of plant. While a transgenic DNA construct can comprise any segmentof DNA that is heterologous to the insertion site, in preferred aspectsof the invention the transgenic DNA construct will be designed toprovide a specific function, e.g., suppress or over express anectoapyrase protein. Useful transgenic DNA constructs may include generegulatory segment operably linked to the protein coding segment orantisense segments. Non-limiting examples of gene regulatory segmentinclude: promoter elements, enhancers, silencers, introns anduntranslated regions.

As used herein, the term “transformation” refers to a method ofintroducing a transgenic DNA construct into a plant genome and caninclude any of the well-known and demonstrated methods including:electroporation (e.g., U.S. Pat. No. 5,384,253); microprojectilebombardment (e.g., U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;6,160,208; 6,399,861 and 6,403,865); Agrobacterium mediatedtransformation (e.g., U.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616;5,981,840 and 6,384,301); and protoplast transformation (e.g., U.S. Pat.No. 5,508,184), relevant portions of each are incorporated herein byreference.

As used herein, the term “tissue from a parental cotton line” refers totissue that is specifically adapted for a selected method oftransformation and can include cell culture or embryonic callus.

Nucleic acid molecules of the present invention may be used intransformation, e.g., integrating exogenous genetic material into aplant cell and the plant cell regenerated into a whole plant. As usedherein, an “exogenous coding region” or “selected coding region” refersto a coding region not normally found in the host genome in an identicalcontext. By “exogenous coding region”, it is meant that the codingregion may be isolated from a different species than that of the hostgenome, or alternatively, isolated from the host genome, but is operablylinked to one or more regulatory regions which differ from those foundin the unaltered, native gene. An exogenous nucleic acid molecule canhave a naturally occurring or non-naturally occurring nucleotidesequence. One skilled in the art understands that an exogenous nucleicacid molecule can be a nucleic acid molecule derived from a different orthe same species into which it is introduced. Such exogenous geneticmaterial may be transferred into either monocotyledons and dicotyledonsincluding but not limited to the plants, soy, cotton, canola, maize,wheat and rice.

Exogenous genetic material that increase or decrease the expression ofectoapyrases in plants may be transferred into a plant cell by the useof a DNA vector or construct designed for such a purpose. Vectors havebeen engineered for transformation of constructs comprising one or morenucleic acid molecules into plant genomes. Vectors have been designed toreplicate in both E. coli and A. tumefaciens and have all of thefeatures required for transferring large inserts of DNA into plantchromosomes. Exogenous genetic material may be transferred into a hostcell by the use of a DNA vector or construct designed for such apurpose.

To suppress ectoapyrase protein gene expression, the heterologous DNAcan be designed to produce a gene silencing effect, e.g., by anantisense or RNAi mechanism. Anti-sense suppression of genes in plantsby introducing by transformation of a construct comprising DNA of thegene of interest in an anti-sense orientation is disclosed in U.S. Pat.Nos. 5,107,065; 5,453,566; 5,759,829; 5,874,269; 5,922,602; 5,973,226;6,005,167; relevant portion of all of which are incorporated herein byreference. Interfering RNA suppression of genes in a plant byintroducing by transformation of a construct that includes DNA encodinga small (commonly less than 30 base pairs) double-stranded piece of RNAmatching the RNA encoded by the gene of interest is disclosed in U.S.Pat. Nos. 5,190,931; 5,272,065; 5,268,149, relevant portion of which areincorporated herein by reference.

Vectors that may be used for plant transformation may include, forexample, Geminiviruses, plasmids, cosmids, YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes), PACs (plantartificial chromosomes), or any other suitable cloning system. A varietyof plant viruses that can be employed as vectors are known in the artand include cauliflower mosaic virus (CaMV), geminivirus, brome mosaicvirus, and tobacco mosaic virus. Particularly useful for transformationare expression cassettes which have been isolated from such vectors. DNAsegments used for transforming plant cells will, of course, generallycomprise the cDNA, gene or genes which one desires to introduced intoand have expressed in the host cells. These DNA segments can furtherinclude structures such as promoters, enhancers, 3′ untranslated regions(such as polyadenylation sites), polylinkers, or even regulatory genesas desired.

Examples of plant viruses suitable for delivery of the ectopyrasemodulators of the present invention include, e.g., wheat dwarf virus,maize streak virus, tobacco yellow dwarf virus, tomato golden mosaicvirus, abutilon mosaic virus, cassaya mosaic virus, beet curly topvirus, bean dwarf mosaic virus, bean golden mosaic virus, chlorisstriate mosaic virus, digitaria streak virus, miscanthus streak virus,maize streak virus, panicum streak virus, potato yellow mosaic virus,squash leaf curl virus, sugarcane streak virus, tomato golden mosaicvirus, tomato leaf curl virus, tomato mottle virus, tobacco yellow dwarfvirus, tomato yellow leaf curl virus, African cassaya mosaic virus, andthe bean yellow dwarf virus.

A number of promoters that are active in plant cells have been describedin the literature. Such promoters would include but are not limited to:nopaline synthase (NOS) and octopine synthase (OCS) promoters that arecarried on tumor-inducing plasmids of Agrobacterium tumefaciens, thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Sand 35S promoters and the figwort mosaic virus (FMV) 35S promoter, theenhanced CaMV35S promoter (e35S), the light-inducible promoter from thesmall subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a veryabundant plant polypeptide). All of these promoters have been used tocreate various types of DNA constructs that have been expressed inplants. See, for example PCT publication WO 84/02913, relevant portionsincorporated herein by reference.

Once the appropriate plant cells are produced that includes theectoapyrase gene modulator, the nucleotide sequences of interest can beintroduced into the plant cells by any method known in the art to obtaingenetically modified plants, plant cells, plant tissue, and seed.Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection,electroporation, Agrobacterium-mediated transformation, direct genetransfer, and ballistic particle acceleration.

Various types of plant tissue can be used for transformation such asembryo cells, meristematic cells, leaf cells, or callus cells derivedfrom embryo, leaf or meristematic cells, however, anytransformation-competent cell or tissue can be used. Various methods forincreasing transformation frequency are disclosed in WO 99/61619; WO00/17364; WO 00/28058; WO 00/37645; U.S. Ser. No. 09/496,444; WO00/50614; US 01/44038; and WO 02/04649, relevant portions incorporatedherein by reference. Once the DNA sequence of interest has beenintroduced into tissue from the plant, transformed cells are selectedand transgenic plants regenerated using methods well known in the art.

Transformed plant cells that are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of plants (e.g., maize)typically produces shoots within two to four weeks and these transformedshoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. The regeneration of plants from either singleplant protoplasts or various explants is well known in the art. See, forexample, Methods for Plant Molecular Biology, A. Weissbach and H.Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988),relevant portions incorporated herein by reference.

Certain vectors may be constructed and employed to specifically targetthe ectoapyrase gene modulation construct within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. Specific targeting can be accomplished by joining a DNAsequence encoding a transit or signal peptide sequence to the codingsequence of a particular gene. An intracellular targeting DNA sequencemay be operably linked 5′ or 3′ to the coding sequence depending on theparticular targeting sequence, e.g., a signal peptide that transportsthe protein to a particular intracellular, or extracellular destination,respectively, and will then be post-translationally removed.

During selection, several antibiotic resistance markers may be used toselect (positively or negatively) the constructs that over or underexpress ectoapyrases for use with the present invention, including genesthat confer resistance to: kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) as well as herbicides such as glufosinate(bar orpat) and glyphosate (EPSPS).

Characterization of cotton apyrase (GhAPY1). The deduced amino acidsequence of GhAPY1 is 471 amino acids in length and possesses all fourof the characteristic apyrase conserved regions. An alignment of thededuced amino acid sequence of the coding regions of GhAPY1, AtAPY1 andAtAPY2 shows significant sequence similarity with GhAPY1 and AtAPY1sharing 67% identity and GhAPY1 and AtAPY2 sharing 68% identity. Withinthe apyrase conserved regions, however, GhAPY1 and AtAPY1 share 97.5%identity, while GhAPY1 and AtAPY2 share 92.5% identity. A query of thepredicted full cotton apyrase amino acid sequence using the SignalP 3.0Server suggests a high probability that there is an uncleavable signalpeptide between residues 20 and 40 (Bendtsen et al., 2004).

Expression of GhAPY1 correlates with rapid phase of fiber elongation.Quantitative RT-PCR analysis of epidermis (fiber) and ovules tissue overa time course during early fiber growth and development shows thatGhAPY1 transcript is mainly found in growing cotton fibers (FIG. 1A).GhAPY1 message first appears in fibers at 2 dpa and then levelsdramatically increase showing a peak at 5 dpa. At 7 dpa message levelsare lower than at 5 dpa but there is still substantial GhAPY1 expressionat this time point. Immunoblot analysis using anti-APY1 and APY2antibodies shows that there is a cross-reactive protein in 7 dpa fibersamples with a similar molecular weight as the Arabidopsis apyrase (FIG.1B).

Apyrase inhibitors and anti-apyrase antibodies inhibit fiber elongation.In order to test whether apyrase activity is important during cottonfiber growth we applied varying concentrations of two different chemicalapyrase inhibitors, apyrase inhibitor 4 and NGXT191, to cotton ovulecultures at 3 dpa and 5 dpa. It was found that application of all threeconcentrations of both inhibitors resulted in statistically significantinhibition of fiber growth when cotton fibers were measured at 7 dpa(FIG. 2A). There is clearly a dose-dependent effect, and the effects ateach dosage are statistically significant. Apyrase inhibitor treatmentsalso resulted in statistically significant inhibition of cotton fibergrowth when fiber lengths were measured at 5 dpa and 19 dpa (data notshown). The level of inhibition increased with increasing concentrationsof both inhibitors. Next when PPADS, an inhibitor of animalpurinoceptors, was tested, it could block the 40% inhibition of fibergrowth caused by application of the highest concentration of apyraseinhibitors. Growth was returned to control levels when 250 μM PPADS wasincluded in the media (FIG. 2B). In order to determine if the observedgrowth effects were indeed due to inhibition of apyrase activity wemeasured the amount of ATP found in the media outside of the fibers andfound that indeed the inhibitor treatments increased the amount ofdetectable ATP outside of the growing fibers from the control level of330 nM (FIG. 2C). As expected increasing the amount of the inhibitorused resulted in higher levels of ATP measured outside of the growingfibers. Application of the highest concentration of inhibitors NGXT191and 4 caused a 2.1-fold and 3.2-fold increase in ATP levels,respectively.

Next, the effects of applying to the cotton ovule cultures polyclonalantibodies raised against Arabidopsis APY1 and APY2 were tested anddemonstrated inhibition of apyrase activity (Wu et al., 2007). It wasfound that treatment with immune sera led to statistically significantinhibition of fiber growth while pre-immune sera had no effect on growth(FIG. 3A). It was also observed that a 3.3 fold and 5.3-fold increase inthe level of ATP in the culture medium was detectable after applicationof the lower and higher antibody concentrations, respectively (FIG. 3B).

Application of high levels of ATPγS and ADPβS inhibit fiber elongation.The results from the inhibitor and antibody treatments suggest thataccumulation of ATP in the fiber ECM causes inhibition of fiberelongation. In order to directly test this hypothesis, we applied poorlyhydrolysable nucleotides to ovule cell cultures. It was found that at 7dpa application of 150 μM ATPγS and ADPβS did indeed inhibit growth andthat application of 200 μM AMPS, a closely related molecule but not apurinoceptor agonist, had no effect on growth (FIG. 4A, B).Statistically significant levels of inhibition of cotton fiber growth at5 dpa by application of high concentrations of 150 μM ATPγS were found.Furthermore, the effects of these high concentrations of ATPγS and ADPβSwere reversed by inclusion of PPADS and Adenosine, two knownpurinoceptor antagonists.

Application of low levels of ATPγS and ADPβS promote fiber elongation.Representative images show that in the presence of 30 μM ATPγS and ADPβScotton fibers grow noticeably longer by 7 dpa and that this promotion ofgrowth can be blocked by the addition of PPADS and adenosine (FIG. 5A).Quantification of these growth changes indicate that these changes arestatistically significant and that 30 μM ATPγS results in a 46% increasein average fiber length and 30 μM ADPβS causes a 44% increase in averagefiber length (FIG. 5B). Also observed were statistically significantlevels of promotion of cotton fiber growth at 5 dpa by 30 μM ATPγS and astatistically significant increase in the growth rate between 5 dpa and7 dpa (data not shown). Application of both ATPγS and ADPβS results in abiphasic dose response curve for fiber growth with the threshold forpromotion of growth somewhere between 10 and 30 μM and the threshold forinhibition of growth somewhere between 125 and 150 μM (FIG. 6).Application of AMPS, which cannot activate purinoceptors in animalcells, has no effect on fiber growth.

Genetic and biochemical approaches have been used to show that twoectoapyrases in Arabidopsis, AtAPY1 and AtAPY2 are critically needed forgrowth. For example, Arabidopsis double knockout mutants (apy1apy2) aresevere dwarfs and inhibition of ectoapyrase activity with eitherchemical inhibitors or specific antibodies leads to inhibition of growth(Wolf et al., 2007; Wu et al., 2007). In addition the expression ofthese two apyrases is found in actively growing tissues (Wolf et al.,2007; Wu et al., 2007). Consistent with this result is the observationthat higher levels of eATP are found in the extracellular matrix ofactively growing tissues in Medicago truncatula (Kim et al., 2006). Thiscorrelation of apyrase expression and localization of eATP in growingcells suggests that it is important to regulate the eATP signal duringgrowth. In this study we show that message levels of the cotton fiberapyrase GhAPY1, which has high sequence similarity to AtAPY1 and AtAPY2,is up-regulated during the rapid phase of fiber elongation.

Motif analysis indicates that GhApy1 contains a putative uncleavablesignal peptide located near its N-terminus. This structure could anchorGhAPY1 to the membrane at its N-terminus and orient the protein domainsresponsible for enzyme activity out into the extracellular matrix,allowing it to function as an ectoapyrase like its close homologuesAtAPY1 and AtAPY2. Consistent with this interpretation, suppression offiber apyrase activity by selective inhibitors leads to an increase inthe concentration of the eATP associated with growing cotton fibers. Thetwo chemical apyrase inhibitors tested have been well characterized(Windsor et al, 2002; Windsor et al., 2003), and have been shown toinhibit pollen tube germination and elongation (Wu et al., 2007). Theyalso inhibit fiber elongation in parallel with the rise in [eATP]. Bothof these inhibitors are sufficiently hydrophobic to cross the plasmamembrane and target apyrases inside the cell, and this could also affectfiber growth. However the chemical inhibitor effects are replicated byanti-apyrase antibodies, which are large proteins that are unlikely tocross the plasma membrane. Our finding that treatment of the growingfibers with anti-apyrase antibodies also resulted in inhibition of fibergrowth and accumulation of eATP strongly supports the hypothesis thatectoapyrase activity is critical for fiber elongation. The antibodiesand apyrase inhibitors would likely inhibit any ectoapyrase expressed incotton fibers, and so, although GhAPY1 expression correlates closelywith fiber growth, it may not be the only ectoapyrase that regulatescotton fiber growth.

The rise in [eATP] that results from suppressing ectoapyrase activitycould be the signal that leads to the inhibition of fiber growth. Thishypothesis is supported by our results showing that application ofmicromolar concentrations of poorly hydrolysable nucleotides can alsoinhibit fiber growth. Because cotton fiber elongation is regulated bymicromolar [eATP] and because antagonists of animal purinoceptors blockthese growth responses, we predict that the metabolic pathway that linkseATP to growth changes in cotton begins in plants as in animals with thebinding of eATP to a plasma membrane receptor. Thus far the plant eATPreceptor has not been found, but recently eATP receptors have beenidentification in the slime mold Dictyostelium and in the green algae(Fountain et al., 2007, 2008).

Results obtained in Arabidopsis thus far indicates the effects of eATPare transduced by an initial increase in the concentration of cytosoliccalcium [Ca^(2+]) _(cyt), followed by the activation of calcium-bindingproteins. Two growth-affecting metabolic steps downstream of (anddependent on) the calcium signal in plants is an increased production ofreactive oxygen species (ROS) and an increased production of ethylene.Recent results in cotton implicate ROS (Li et al., 2007) and ethylene(Shi et al., 2006) as key mediators of cotton fiber growth. InArabidopsis genetically blocking ROS production blocks eATP effects onwound signaling, and blocking ethylene production blocks eATP effects ongrowth inhibition (Butterfield, 2007). Moreover, applied ATP induces ACCsynthase expression in a calcium-dependent way (Jeter et al., 2004). Weare currently testing whether eATP induced changes in cotton fiberelongation are linked to increased production of ROS and/or an increasedproduction of ethylene in cotton fibers, both of which are dependent onextracellular calcium.

There are other hormone-mediated signaling pathways that could also beinteracting with the eATP signal to regulate fiber growth. For example,apyrases and eATP have been implicated in the inhibition of thetransport of the growth hormone, auxin (Tang et al., 2003). eATP hasalso been shown to promote the production of nitric oxide (NO) in tomatocell suspensions and during wound responses in the green algae,Dasycladus vermicularis (Foresi et al., 2007; Torres et al., in press).NO also appears to play an important role during plant growth (Pagnusatet al., 2004). It is interesting to find that eATP/apyrase signalingregulates growth in pollen tubes which grow via polar tip growth as wellas cotton fibers which grow via diffuse growth as there are clearcytological differences during these two types of growth (Tiwari andWilkins, 1995). In both cases growth would be expected to be accompaniedby fusion of secretory vesicles the possible release of ATP which couldthen act as a feedback signal to regulate growth. Genetic studies willbe the next step toward further clarifying the precise function ofGhApy1 and the role of extracellular nucleotide signaling during cottonfiber growth.

Plant Materials. Upland cotton (TM-1) was grown in a soil mixture in afully automated green house. Flowers were picked and used for in vitroovule culture studies. In studies using RNA extraction, 2- to 7-DPAovules were harvested and immediately frozen using liquid nitrogen andthen stored in a −80° C. refrigerator until they were analyzed.

Cloning of GhAPY1. Cotton EST TC59992, a putative apyrase full-lengthconsensus sequence assembled from partial transcripts from severallibraries (including G. arboreum, G. raimondii, and G. hirsutum) wasgenerously supplied by Andrew Woodward in the C. Chen lab at theUniversity of Texas. This sequence was used to design PCR primers withthe following sequences: GhAPY1F (5′ATGATCAAGCGTTCAATGGCG3′) (SEQ IDNO.: 1) and GhAPY1R (5′CCTCATTGCTGATACAGCTTCGATGGC3′) (SEQ ID NO.: 2),which were used to amplify RNA message from whole cotton leaf RNA.Cotton leaf RNA was isolated using the Sigma Spectrum Plant Total RNAKit kit, and first-strand cDNA was synthesized using RT-PCR. This cDNAwas used as template for Roche Expand Hi-Fidelity Taq DNA polymerase toamplify putative cotton apyrase. Amplified message was cloned into theexpression vector pTrcHis2 using Invitrogen pTrcHis and pTrcHis2 TOPO TAExpression kit. The vector used in this kit adds both Myc and6×-Histidine tags to the C-terminus of the cloned sequence. InvitrogenTop 10 chemically competent E. coli cells were then transformed withthis construct. The sequence of the putative transformants wereconfirmed at the University of Texas ICMB Core DNA facility bysequencing with AB 3130 and AB 3730 DNA Analyzers.

Laser capture microdissection (LCM) and RNA preparation. For the youngovule tissues such as the ovules at −2 DPA, 0 DPA, and 2 DPA, lasercapture microdissection and antisense RNA amplification were necessarybecause the fiber initials (or primordial cells in the epidermis) duringfiber initiation are only a small portion of the ovule cells which makestechnical difficulties on tissue preparation. Cotton ovules collected at−2 DPA, 0 DPA, and 2 DPA were fixed in a fixative (3:1 ethanol:aceticacid) for 10 min and vacuum infiltrated on ice for 20 min. Theinfiltrated tissues were incubated at 4° C. for 1 hour with rotation.The vacuum infiltration and incubation process were repeated twice withfresh fixative. Tissues infiltrated with 10 ml of 10% sucrose for 15min, rotated overnight at 4° C. and repeated with 15% sucrose forcryoprotection. The fixed ovules were embedded with Tissue-Tek® OptimalCutting Temperature (O.C.T.) (Sakura Finetek U.S.A., Torrance, Calif.)in cryo-mold. The embedded ovules were freezed immediately in liquidnitrogen and stored at −80° C.

Cryosectioning was performed with a Leica Cryostat (Leica Microsystems,Bannockburn, Ill.) in the microscopy facility at the University of Texasat Austin. The block was equilibrated at −20° C. for 1 hour andcryosectioned at 10 μm. The slides with cryosectioned ovules weredehydrated with a series of ethanol (70%, 95%, and 100%) form 2 min eachon ice and transferred to histoclear (National Diagnostics, Atlanta,Ga.).

The PALM laser capture system (P.A.L.M. Microlaser Technologies AG Inc.,Bernried, Germany) was used for laser capture microdissection.Individual fiber initials (−2 DPA and 0 DPA) or epidermal cells (2 DPA)were catapulted and then 45 μl of RNALater (Ambion, Austin, Tex.) wasadded. The antisense RNAs were processed from the captured cells usingMessageAmp II aRNA amplification kit (Ambion, Austin, Tex.). Cottonovules at 5 DPA and 7 DPA were processed with Spectrum Plant RNA kit(Sigma, St. Louis, Mo.) without laser capture microdissection.

Quantitative RT-PCR (qRT-PCR) analysis. First strand cDNA synthesis wasperformed using aRNA (for tissues at −2 DPA, 0 DPA, and 2 DPA) or totalRNA (for tissues at 5 DPA and 7 DPA) and SuperScript II (Invitrogen,Carlsbad, Calif.). For the transcript amplification, gene-specificprimers (Forward: 5′-ATC CAC AGG CTG CTG CAA AT-3′ (SEQ ID NO.: 3),Reverse: 5′-AAT GCC CTC AGA CCA GCA GTT-3′ (SEQ ID NO.: 4)) weredesigned using Primer Express version 2.0 software (Applied Biosystems,Foster City, Calif.). The qRT-PCR reaction was carried out in a finalvolume of 20 μl containing 10 μl SYBR Green PCR master mix (AppliedBiosystems, Foster City, Calif.), 1 μMforward and reverse primers, and0.1 μM cDNA probe in a ABI7500 Real-Time PCR system (Applied Biosystems,Foster City, Calif.). Cotton HISTONE3 (AF024716) was used to normalizethe amount of gene-specific RT-PCR products (Wang et al., 2004). Allreactions were performed in three replications and the amplificationdata were analyzed using ABI7500 SDS software (version 1.2.2) and thefold changes were calculated using the standard in each reaction.

Immunoblot analysis. At 0 and 7 DPA of in vitro ovule culture, cottonovules were harvested and samples were placed in 1.5 ml centrifuge tubesthen frozen with liquid nitrogen. Frozen samples were then ground at 4°C. using a micro mortar in the presence of a buffer containing 45 mMTris, 150 mM NaCl, 1.2 mM EGTA, 5 mM DTT and protease inhibitors.Samples were then aliquoted for loading and boiled in the presence ofSDS sample buffer for 3 min. After boiling, samples were centrifuged for30 s at RT. Proteins were then separated via 10% SDS-PAGE, transferredto 0.45 μm nitrocellulose membranes (Schleicher & Schuell), and blockedfor 2 hrs with 1% dry milk in PBS pH 7.5 (Blotto). The membrane was thenincubated overnight at 4° C. with protein A-sepharose purified anti-Apylantibody diluted 1:250 with 1% Blotto. After three washes in 1% Blotto,the membrane was incubated for 1 hr at room temperature withaffinity-purified anti-guinea pig IgG (goat) coupled to an 800-nmfluorochrome diluted 1:10,000 (Rockland IRDye 800CW). After three washeswith Blotto, the fluorochrome signals were detected and analyzed usingthe Odyssey infrared imaging system (LI-COR Biosciences).

In Vitro Ovule Culture. Cotton bolls were harvested within 24 hours offlower opening, 1-DPA, and all petals bracts, and sepals were removed.All collected cotton bolls were used within 24 hours of originalharvest. If the bolls were not used immediately they were stored in a 4°C. refrigerator until ready for use. For sterilization, bolls weredropped into an 85% ethanol solution for at least 30 seconds but notmore than two minutes and then removed with forceps. The excess ethanolwas shaken off and the boll was passed through a flame until theresidual ethanol was burned off. Ovules were then excised and placed in10 mL of BT Cotton Media cell culture (Caisson Laboratories, Inc., NorthLogan, Utah, USA)+5.0 μM IAA and 0.5 μM GA₃ (Sigma, St. Louis, Mo., USA)hormone solution, final concentration, in 60×15 mm Petri dishes and werethen wrapped in Milli Wrap (Millipore Corporation, Bedford, Mass.).Twelve to 16 cotton ovules were placed into their respective Petridishes and incubated with nucleotides, inhibitors, antibody serum or acombination of these at specified concentrations and were cultured at32° C. in the dark without agitation. To facilitate combing of thecultured ovules, ovules were placed in a 75% ethanol solution for 15minutes and then taken out to be combed. Fiber lengths were measuredmanually with a dissecting microscope after laying the ovule on its sideand combing the fiber cells away from the ovule with forceps anddissecting probe.

In Vitro Cotton Ovule Culture Treatments. For studies testing theeffects of apyrase inhibitors on cotton fiber growth, the inhibitors(NGXT191 and AI#4, both at 2.5 mg/mL), were dissolved in dimethylsulfoxide and then applied to Cotton Media cell culture at 3 and 5 DPAafter the introduction of cotton ovules to the media. For experimentstesting the effects of apyrase antibodies on cotton fiber growth,apyrase antibodies were applied to Cotton Media cell culture at 3 and 5DPA after the introduction of cotton ovules in the appropriate amounts.For experiments testing the effects of applying nucleotides duringcotton fiber growth, various concentrations of ATPγS, ADPβS, and AMP(Sigma-Aldrich, Inc., St. Louis, Mo., USA) were added to Cotton Mediacell culture at 5 DPA after the introduction of cotton ovules in theappropriate amounts. All nucleotides were made into 50 mM stocksdissolved in de-ionized water and kept at −20° C. while not in use. Theinhibitor stocks were stored at −20° C. while not in use. Forexperiments testing the effects of P2-receptor antagonists, theantagonists (PPADS and Adenosine, stocks at 50 mM) were dissolved inde-ionized water and then applied to Cotton Media cell culture at 3 and5 DPA for inhibitor and antibody experiments and 5 DPA for appliednucleotide experiments. The antagonists stocks were stored at −20° C.while not in use (Sigma-Aldrich, Inc., St. Louis, Mo., USA). Theethylene inhibitor (S)-trans-2-Amino-4-(2-aminoethoxy)-3-butenoic acidhydrochloride (Sigma-Aldrich, Inc., St. Louis, Mo., USA) (AVG, stock at50 mM) was dissolved in de-ionized water and then applied to CottonMedia cell culture at 5 DPA. The production of the Arabidopsis anti-APY1and APY2 antibodies used is described by Steinebrunner et al. (2003).The crude immune and pre-immune sera were purified using proteinA-sepharose following the protocol described by Martin et al. (1982)with the slight modification that the buffers used were azide-free.Bio-rad assay was used to determine that the concentration of the immuneserum and pre-immune serum which was 0.46 μg/mL and 0.3 μg/mL,respectively.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1. A method of modulating plant fiber growth comprising: contacting a plant cell with one or more extracellular exogenous nucleotides selected from di-nucleotides, tri-nucleotides, or poorly-hydrolyzable nucleotides at a concentration that modulates growth of one or more cotton fibers.
 2. The method of claim 1, wherein the one or more poorly-hydrolyzable nucleotides comprise thio, methylene, amide or methyl-modified ATP, ADP, UTP, UDP, CTP, CDP, TTP, TDP, GTP, GDP, dATP, dADP, dUTP, dUDP, dCTP, dCDP, dTTP, dTDP, dGTP, dGDP, analogues and combinations thereof.
 3. The method of claim 1, wherein the one or more extracellular nucleotides are provided in a concentration that increases growth of the one or more cotton fibers.
 4. The method of claim 1, wherein the one or more extracellular nucleotides are provided in a concentration that decreases growth of the one or more cotton fibers.
 5. The method of claim 1, wherein the one or more extracellular nucleotides are provided at a concentration between 1 μM and 100 μM to increase the growth of the one or more cotton fibers.
 6. The method of claim 1, wherein the one or more extracellular nucleotides are provided at a concentration between 125 μM and 200 μM to decrease the growth of the one or more cotton fibers.
 7. The method of claim 1, wherein the one or more extracellular nucleotides are provided at a concentration between 10 μM and 75 μM to increase the growth of the one or more cotton fibers.
 8. The method of claim 1, wherein the one or more extracellular nucleotides are provided at a concentration of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μM that increases the growth of the one or more cotton fibers.
 9. The method of claim 1, wherein the one or more extracellular nucleotides are provided at a concentration of 125 μM or greater to decrease the growth of the one or more cotton fibers.
 10. The method of claim 1, wherein the one or more extracellular nucleotides alter the activity of one or more ectoapyrase enzymes.
 11. A method of modulating plant fiber growth comprising: contacting a plant cell with one or more modulators of ectoapyrase gene transcription, wherein the modulation alters the length of one or more cotton fibers.
 12. The method of claim 11, wherein the one or more modulators of ectoapyrase gene transcription alters ectoapyrase gene transcription are selected from anti-sense or siRNA gene inhibitors.
 13. The method of claim 11, wherein the one or more modulators of ectoapyrase gene transcription are antagonists of ectoapyrase gene transcription to decrease fiber growth.
 14. The method of claim 11, wherein the one or more modulators of ectoapyrase gene transcription are agonists of ectoapyrase gene transcription to increase fiber growth.
 15. A recombinant plant comprising a plant cell that has increased expression of ectoapyrases that increases cotton fiber growth.
 16. A method of modulating plant fiber growth comprising: contacting a plant cell with one or more inhibitors of ectoapyrase activity comprising an anti-ectoapyrase antibody or fragments thereof, wherein the inhibition decreases the length of one or more cotton fibers.
 17. A composition that modulates the length of one or more cotton fibers in a plant comprising at least one of an extracellular exogenous nucleotides selected from di-nucleotides, tri-nucleotides, or poorly-hydrolyzable nucleotides, a modulator of ectoapyrase gene transcription, an anti-ectoapyrase antibody or fragments thereof, at a concentration sufficient to modulate growth of one or more cotton fibers.
 18. The composition of claim 17, wherein the one or more poorly-hydrolyzable nucleotides comprise thio, methylene, amide or methyl-modified ATP, ADP, UTP, UDP, CTP, CDP, TTP, TDP, GTP, GDP, dATP, dADP, dUTP, dUDP, dCTP, dCDP, dTTP, dTDP, dGTP, dGDP, ATPγS, ADPβS, and analogues and combinations thereof.
 19. The composition of claim 17, wherein the one or more extracellular nucleotides are provided in a concentration that increases growth of the one or more cotton fibers.
 20. The composition of claim 17, wherein the one or more extracellular nucleotides are provided in a concentration that decreases growth of the one or more cotton fibers.
 21. The composition of claim 17, wherein the one or more extracellular nucleotides are provided at a concentration between 1 μM and 100 μM to increase the growth of the one or more cotton fibers.
 22. The composition of claim 17, wherein the one or more extracellular nucleotides are provided at a concentration between 125 μM and 200 μM to decrease the growth of the one or more cotton fibers.
 23. The composition of claim 17, wherein the one or more extracellular nucleotides are provided at a concentration between 10 μM and 75 μM to increase the growth of the one or more cotton fibers.
 24. The composition of claim 17, wherein the one or more extracellular nucleotides are provided at a concentration of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μM that increases the growth of the one or more cotton fibers.
 25. The composition of claim 17, wherein the one or more extracellular nucleotides are provided at a concentration between 150 μM and 200 μM to decrease the growth of the one or more cotton fibers.
 26. The composition of claim 17, wherein the one or more extracellular nucleotides alter the activity of one or more ectoapyrase enzymes.
 27. A recombinant plant exhibiting increased cotton fiber growth as compared to the corresponding wild-type plant, wherein the recombinant plant comprises a recombinant nucleic acid encoding an ectoapyrase gene inhibitor operably associated with a regulatory sequence.
 28. The recombinant plant of claim 27, wherein the regulatory sequence is a promoter, a constitutive promoter or an inducible promoter.
 29. The recombinant plant of claim 27, wherein the nucleic acid is contained within a T-DNA derived vector.
 30. Recombinant plant tissue derived from the recombinant plant of claim
 27. 31. A recombinant seed derived from the recombinant plant of claim
 30. 32. A method of making recombinant plant exhibiting increased cotton fiber growth as compared to the corresponding wild-type plant comprising: contacting plant cells with a nucleic acid encoding an inhibitor of an ectoapyrase, wherein the nucleic acid is operably associated with a regulatory sequence to obtain transformed plant cells; producing plants from the transformed plant cells; and selecting a plant exhibiting the increased cotton fiber growth and yield.
 33. A recombinant plant tissue derived from the recombinant plant of claim 31
 34. The recombinant seed derived from the recombinant plant of claim
 33. 